Novel materials for dental and biomedical application

ABSTRACT

The invention provides novel dental enamel inspired materials for biomedical and dental applications. The materials are apatite-like calcium phosphate complexes and may comprise apatite, octacalcium phosphate crystals, or mixtures thereof. In one embodiment, the materials (calcium phosphate coatings) are mixtures of crystals of apatite and its precursor, octacalcium phosphate, nucleated on a titanium surface. They are prepared using a chemical process leading to the formation of biological apatite which is similar to that found in natural bone and teeth. In one embodiment, the materials are prepared by placing a titanium substrate in a supersaturated calcifyng solution containing native or purified recombinant amelogenins. The presence of the amelogenins modulates apatite crystal growth to mimic in vivo apatite crystal formation. Applications for the materials include, without limitation, dental tissue (enamel, dentin, cementum) replacement, orthopeadic implants for bone repair, and coatings for improving the biocompatibility and bone regeneration capability of currently available implants or medical devices made of metallic, polymeric, ceramic or composite materials.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) of U.S.Provisional Application Ser. No. 60/358,157, filed Feb. 20, 2002,entitled “Novel Materials for Dental and Biomedical Application.” Theapplication is incorporated herein by this reference.

STATEMENT AS TO INVENTION RIGHTS UNDER FEDERALLY SPONSORED RESEARCH

This work was supported by NICDR research grants DE12350 and DE13414.The United States government may have certain rights in the inventionsdisclosed herein.

BACKGROUND

Biological implants, such as joint and dental prostheses, usually mustbe permanently affixed or anchored within bone. In some instances it isacceptable to use a bone cement to affix the prosthesis within bone. Inthe case of many joint prostheses, however, it is now more common toaffix the joint prosthesis by encouraging natural bone growth in andaround the prosthesis. Bone-to-implant interfaces that result fromnatural bone ingrowth tend to be stronger over time and more permanentthan are bone cement-prosthesis bonds.

Optimal bone ingrowth requires that natural bone grow into and aroundthe prosthesis to be implanted. Bone ingrowth and bone or dentalprosthesis fixation can be enhanced by providing irregular beaded orporous surfaces on the implant. Although various materials, includingtitanium alloys, are biocompatible, they are not necessarily bioactivebecause they can neither conduct bone formation nor form chemical bondswith bone. Thus, enhanced fixation of implants within bone can beattained by coating the implant with a bioactive mineralized and/orceramic material. Such coatings have been shown to encourage more rapidbone ingrowth in and around the prosthesis.

Calcium phosphate ceramics, especially hydroxyapatite, have been shownto conduct bone formation. Hydroxyapatite ceramic has been successfullyapplied as a coating on cementless metallic implants to achieve quickand strong fixation. Thermal plasma spraying is one of the more commonmethods used to produce hydroxyapatite coatings. However, the resultingplasma-sprayed hydroxyapatite coating is of relatively low density andis not uniform in structure or composition. The adhesion between thecoating and substrate is generally not very strong, especially afterlong term exposure within the body. The generation of hard ceramicparticles, resulting from the degradation of thermal plasma sprayedcoating, and coating delamination, are major concerns. Also, implants orother polymers with porous structures or complex surfaces are difficultto coat uniformly using line-of-sight temperature plasma spraying.

A general overview of orthopedic implantable materials is given inDamien, Christopher J., and Parsons, Russell J., 1991, Journal ofApplied Biomaterials, v2, 187-208. Information related to attempts toaddress these problems can be found, e.g., in U.S. Pat. Nos. 6,139,585;6,051,272; 5,609,633; and in some of the publications disclosed herein.Each of these references suffers from one or more of the followingdisadvantages: weakness, brittleness or unevenness of coatings, a lackof chondrogenic or osteogenic activities, inability to promote theregeneration of periodontal tissues, and the problem of contaminationwith components which may cause severe immunological reactions.

Thus, a need exists for the production of improved enamel inspiredmaterials which are appropriate for biomedical and dental applicationsand which overcome the problems discussed above.

SUMMARY

The invention provides an improved method for synthesizing coatedimplantable articles suitable for biomedical and dental applications. Inone embodiment, the method generally comprises the step of contacting animplantable substrate to be coated with a supersaturated calcifyingsolution, where the solution comprises an effective amount of anamelogenin-type protein. Typically, the substrate will be immersed inthe solution under suitable temperature conditions until the desiredamount of an enamel-like biomaterial coats the substrate surface. Thesurface may be coated partially or entirely with the enamel-likebiomaterial, depending on the user's preference. Where the coatingappears, it is chemically bonded to the surface of the substrate.

The method may be applied to a variety of substrates, including metals,ceramics, polymers and silicon. In particular, the method is useful forcoating substrates which are intended for medical implantation, such asbone and dental prostheses. In one embodiment, the substrate may becomposed of a strong biocompatible material, for example, a metal suchas titanium. In other embodiments, metals including titanium alloy,tantalum, tantalum alloy, stainless steel or cobalt chromium alloy arecoated. Other embodiments of the method use well-known biocompatiblematerials such as ultra high molecular weight polyethylene,hydroxyapatite, Bioglass and Glass Ceramic A-W.

Another embodiment of the invention includes a step in which the metalsubstrate is activated for crystal growth by a nanometer-scaled porousoxide layer at the metal's surface. One means of achieving suchactivation is by etching. Many methods of etching are known to thoseskilled in the art. One useful etching method comprises the steps ofcontacting the substrate with an effective amount of acid and aneffective amount of an oxidizing agent.

In one embodiment of the method of the invention, the substrate to becoated is contacted with a supersaturated calcifying solution comprisingcalcium phosphate and a buffer which maintains an approximately neutralpH. Typically, the coating reaction is carried out at any temperaturebetween approximately ambient room temperature and a biologicallyrelevant temperature such as the temperature of the human body (i.e., 37degrees Celsius).

The ionic strength of the supersaturated calcifying solution is betweenapproximately 50 mM and 500 mM. In one embodiment, the ionic strength isbetween 100 and 200 mM.

In another embodiment, the amelogenin-type protein dissolved in thesupersaturated calcifying solution has a function similar to that ofmouse amelogenin rM179. In a related embodiment, the amelogenin-typeprotein comprises the sequence shown in SEQ ID No. 1. Theamelogenin-type protein is typically present at concentrations greaterthan approximately 12.5 μg/ml, including concentrations of 100 μg/ml, orhigher.

In other embodiments of the method, the substrate to be coated isexposed to the calcifying solution for approximately an hour or more.The substrate may be exposed to the solution for 24 hours or longer,depending on the thickness or extent of substrate coating desired.

In certain embodiments of the coating method, additional steps areadded. For example, in one embodiment, before contacting the substratewith the calcifying solution containing the amelogenin-type protein, thesubstrate is first contacted with a supersaturated calcifying solutionwhich is substantially free of any amelogenin-type protein.

A further object of the invention is to provide a coating method whereinagents in addition to the amelogenin-type protein are included in thecalcifying solution or solutions to which the substrate is exposed. Forexample, in some embodiments, therapeutic agents such as antibiotics,growth factors, or anti-inflammatory agents are added to the calcifyingsolution and incorporated into the enamel-like coating.

The amelogenin-type protein of the invention may be incorporated intothe enamel-like coating at varying levels depending on parameters suchas the concentration of the amelogenin-type protein in the calcifyingsolution. In some embodiments, the enamel-like coating comprises between1×10⁻³% and 1% w/v amelogenin-type protein, although the amounts mayvary, for example, to encompass the range from 1×10⁻⁴% and 10% w/vamelogenin-type protein.

Another object of the invention is to provide a method for coating animplantable substrate with an enamel-like biomaterial which comprisessubmicron bundles of elongated apatite crystals with an average aspectratio (length/width) of at least two.

Yet another object of the invention is to provide a method for modifyingthe growth of apatite crystals on an implantable substrate. According toone embodiment of this method, crystal growth modification is achievedby the addition of an effective concentration of an amelogenin-typeprotein to a supersaturated calcifying solution. The substrate on whichcrystals are grown is then be contacted with the calcifying solutionunder suitable conditions until the desired growth of crystals isachieved. Typically, the growth of apatite crystals will be modified toproduce submicron-sized crystals with an average aspect ratio ofapproximately two or greater.

This invention also provides the articles produced by the methodsdescribed herein.

A further object of the invention is to provide an implantable articlewhich comprises a biocompatible substrate coated with an enamel-likebiomaterial. In one variation of this embodiment of the invention, theenamel-like surface coating is chemically bonded to at least a portionof the substrate and comprises apatite crystals and an amelogenin-typeprotein. In a related embodiment, the crystals are less than 1 μm inlength with an average aspect ratio (length to width) of approximatelytwo or greater. In various other embodiments, the crystals whichcomprise the substrate coating contain carbonate or magnesium inaddition to calcium and phosphate, and the calcifying solutions comprisemagnesium, sodium, sulfate, chlorine, carbonate or silicate ions, ormixtures thereof.

In a related embodiment, the enamel-like coating of the inventioncomprises, in addition to amelogenin-type proteins, a therapeutic agentor agents. Such agents include, but are not limited to, growth factorssuch as, bFGF, aFGF, EGF (epidermal growth factor), PDGF(platelet-derived growth factor), IGF (insulin-like growth factor),TGF-.beta. 1 through 3, including the TGF-.beta. superfamily (BMP's,GDF-5, ADMP-1 and dpp); cytokines, such as various interferons,including interferon-alpha, -beta and -gamma, and interleukin-2 and -3;hormones, such as insulin, growth hormone-releasing factor andcalcitonin; non-peptide hormones; antibiotics; anti-cancer agents andchemical agents, such as chemical mimetics of growth factors or growthfactor receptors. In certain embodiments, the therapeutic agents includethose factors, proteinaceous or otherwise, which are found to play arole in the induction or conduction of growth of bone, ligaments,cartilage or other tissues associated with bone or joints, such as forexample, BMP and bFGF.

Yet another object of the invention is to provide a method of enhancingbone ingrowth or soft tissue attachment by implanting an article coatedwith the enamel-like biomaterial of the invention onto a bone surface orsoft tissue. In a related embodiment, the invention provides a methodfor delivering a therapeutic agent comprising implanting the an articlecoated with the enamel-like biomaterial of the invention, and furthercomprising a therapeutic agent, onto a bone surface or soft tissue.

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SEM micrographs of titanium surfaces: (a) untreated, (b) HFetching, (c) HF etching and NaOH immersion. Arrows point tonano-sized-structures in (b) and nano-sized-pores in (c).

FIG. 2 shows XRD (X-ray diffraction) patterns of Ca—P formed onchemically modified titanium via Process I (a-c) at: (a) no protein, (b)100 μg/ml rM 179 and (c) 100 μg/ml BSA and Process II (d-f) at: (d) noprotein, (e) 100 μg/ml BSA and (f) 100 μg/ml rM 179. The patterns (g)and (h) were from samples after 4 h of SCS1 immersion and fromchemically modified titanium, respectively. The diffraction peaks wereassigned according to the standard JCPDS cards (1980, JCPDSInternational Center for Diffraction Data. Powder Diffraction File.Swarthmore, Pa.).

FIG. 3 shows SEM micrographs of the cross section (a) of the Ca—Pcoating formed on the chemically modified titanium (Ti) via Process Iand the morphologies of OCP crystals formed at (b) no protein, (c) 50and (d) 100 μg/ml rM179, (e) 50 and (f) 100 μg/ml BSA.

FIG. 4 shows SEM micrographs of apatite (Ap) crystals initially inducedby chemically modified titanium (Ti) after 4 h SCS1 immersion (a, c) andapatite coatings formed via Process II at (b, d) no protein, (e) 50 and(f) 100 μg/ml rM179, (g) 50 and (h) 100 μg/ml BSA. Images at (c) and (d)are respectively the higher magnifications of (a) and (b).

FIG. 5 shows TEM images (a) of the apatite crystals formed via ProcessII in the presence of 100 μg/ml of rM179. The enlargement of the leftbundle of crystals in (a) was shown in (b) and the corresponding SAEDwas shown in (c). The SAED pattern of apatite was indexed according tothe standard JCPDS cards (1980, JCPDS International Center forDiffraction Data. Powder Diffraction File. Swarthmore, Pa.).

FIG. 6 shows Tapping Mode AFM images of the untreated (as received)Bioglass surface (A) and those after 0.5 h of immersion in SCS1_(rP172)(B) and 4 h of immersion in SCS1_(b) (C) and SCS1_(rP172) (D). Arrowsdenote polishing scratches in A, apatite mineral in C and D; arrowheadsindicate nanosphere assemblies in B.

FIG. 7 shows SEM micrographs of the apatite crystals formed on Bioglasssamples after incubation in PBS for 1 week and subsequent immersion inblank, BSA- and rM179-containing SCS2 for 3 days.

DETAILED DESCRIPTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

Definitions

As used herein, certain terms have the following defined meanings.

As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “an article” includes a plurality ofarticles.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably to refer to polymeric forms of nucleotides of anylength. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes, for example,single-stranded, double-stranded and triple helical molecules, a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A nucleic acid molecule may alsocomprise modified nucleic acid molecules.

The term “peptide” is used in its broadest sense to refer to a compoundof two or more subunit amino acids, amino acid analogs, orpeptidomimetics. The subunits may be linked by peptide bonds. In anotherembodiment, the subunit may be linked by other bonds, e.g. ester, ether,etc. As used herein the term “amino acid” refers to either naturaland/or unnatural or synthetic amino acids, including glycine and boththe D or L optical isomers, and amino acid analogs and peptidomimetics.A peptide of three or more amino acids is commonly called anoligopeptide if the peptide chain is short. If the peptide chain islong, the peptide is commonly called a polypeptide or a protein.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubationtemperatures of about 25° C. to about 37° C.; hybridization bufferconcentrations of about 6×SSC to about 10×SSC; formamide concentrationsof about 0% to about 25%; and wash solutions of about 6×SSC. Examples ofmoderate hybridization conditions include: incubation temperatures ofabout 40° C. to about 50° C.; buffer concentrations of about 9×SSC toabout 2×SSC; formamide concentrations of about 30% to about 50%; andwash solutions of about 5×SSC to about 2×SSC. Examples of highstringency conditions include: incubation temperatures of about 55° C.to about 68° C.; buffer concentrations of about 1×SSC to about 0.1×SSC;formamide concentrations of about 55% to about 75%; and wash solutionsof about 1×SSC, 0.1×SSC, or deionized water. In general, hybridizationincubation times are from 5 minutes to 24 hours, with 1, 2, or morewashing steps, and wash incubation times are about 1, 2, or 15 minutes.SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood thatequivalents of SSC using other buffer systems can be employed.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 80%, 85%,90%, or 95%) of “sequence identity” to another sequence means that, whenaligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. This alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30, section7.7.18, Table 7.7.1. Preferably, default parameters are used foralignment. A preferred alignment program is BLAST, using defaultparameters. In particular, preferred programs are BLASTN and BLASTP,using the following default parameters: Genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

The term “isolated” means separated from constituents, cellular andotherwise, in which the polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, are normally associated with in nature.For example, with respect to a polynucleotide, an isolatedpolynucleotide is one that is separated from the 5′ and 3′ sequenceswith which it is normally associated in the chromosome. As is apparentto those of skill in the art, a non-naturally occurring polynucleotide,peptide, polypeptide, protein, antibody, or fragments thereof, does notrequire “isolation” to distinguish it from its naturally occurringcounterpart. In addition, a “concentrated”, “separated” or “diluted”polynucleotide, peptide, polypeptide, protein, antibody, or fragmentsthereof, is distinguishable from its naturally occurring counterpart inthat the concentration or number of molecules per volume is greater than“concentrated” or less than “separated” than that of its naturallyoccurring counterpart. A polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, which differs from the naturallyoccurring counterpart in its primary sequence or for example, by itsglycosylation pattern, need not be present in its isolated form since itis distinguishable from its naturally occurring counterpart by itsprimary sequence, or alternatively, by another characteristic such asglycosylation pattern. Although not explicitly stated for each of theinventions disclosed herein, it is to be understood that all of theabove embodiments for each of the compositions disclosed below and underthe appropriate conditions, are provided by this invention. Thus, anon-naturally occurring polynucleotide is provided as a separateembodiment from the isolated naturally occurring polynucleotide. Aprotein produced in a bacterial cell is provided as a separateembodiment from the naturally occurring protein isolated from aeucaryotic cell in which it is produced in nature.

A “subject” is a vertebrate, preferably an animal or a mammal, morepreferably a human. Mammals include, but are not limited to, murines,simians, humans, farm animals, sport animals, and pets.

A “control” is an alternative subject or sample used in an experimentfor comparison purpose. A control can be “positive” or “negative”. Forexample, where the purpose of the experiment is to determine acorrelation between concentration of amelogin-type protein and crystalformation, it is generally preferable to use a positive control (asample having a previously determined correlation), and a negativecontrol (a sample lacking amelogin-type protein).

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin REMINGTON'SPHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which may bevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are well known in the art.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

The invention provides a method for coating articles suitable for dentalor prosthetic implantation with an enamel-like biomaterial. The phrase“enamel-like” refers to the similarities to natural enamel, in terms ofstrength and crystal habit, as well as the bioactive properties of thecoating, relative to other synthetic derivatives created without the useof amelogenin-like proteins. The bioactivity of the enamel-like coatingmanifests itself at least partly in the coating's advantageous abilityto promote the ingrovth of natural bone and/or oral tissue around thecoated prosthesis. During the natural growth of bone and teeth, a softextracellular organic matrix serves as a dynamic scaffold to control andfacilitate the formation of highly ordered, remarkably elongatedcarbonated apatite crystals while it is being progressively degraded,leading to a mature enamel composed of more than 95 wt % inorganicminerals (Fincham et al. 1999, J Struct Biol, 122, 320-327). The fusionof natural bone and/or tooth enamel to the coated implants provided bythe present invention will provide the implants with greater strength,durability and overall utility.

Without being bound by this proposed mechanism of action, the bioactiveproperties of the coated inserts produced by the methods of theinvention are the result, at least partially, of the presence ofamelogenin-type proteins in the calcifying solutions used in the method.Amelogenins are the major protein component of the developing dentalenamel, accounting for about 90% of the extracellular organic matrix(Termine et al. 1980, J. Biol. Chem., 255, 9760). The primary structuresof amelogenins are highly conserved across species (Fincham et al. 1997,Dental Enamel—Ciba Foundation Symposium 205, D. J. Chadwick, and G.Cardew (Eds.), John Wiley & Sons, Chichester, 118). The parent orfull-length amelogenin molecule is comprised of the following threeregions: (i) an N-terminal sequence of some 44-45 residues (referred toas TRAP, for Tyrosine-Rich Amelogenin Polypeptide); (ii) a hydrophobiccore sequence of some 100-130 residues enriched in proline, leucine,methionine and glutamine; and (iii) an acidic hydrophilic C-terminalsequence of some 15 residues (Fincham et al. 1997, Dental Enamel—CibaFoundation Symposium 205, D. J. Chadwick, and G. Cardew (Eds.), JohnWiley & Sons, Chichester, 118). The hydrophilic C-terminal of the parentamelogenin is proteolytically cleaved shortly after secretion (Finchamet al. 1996, Connect. Tissue Res., 35, 151). Other amelogenin isoformsin the extracellular matrix include the proteolytic product (TRAP) and afew alternative splice products such as the leucine-rich amelogeninpolypeptide (LRAP). The term “amelogenin-type proteins” refers tomembers of this protein family and their natural or syntheticderivatives, for example, as described herein as having the same orsimilar ability to enhance or modulate crystal formation compared torM179. One skilled in the art will recognize suitable members of thisfamily according to their amino acid sequences and their effects on boneand/or tooth development in vivo and in vitro. For example, amino acidsequences with 40% or more identity to mouse amelogenin are consideredhomologs and are expected to have the similar structures and functions.

LRAP is identical to the full-length amelogenin protein at its twotermini but lacks a large central segment of the protein (Gibson et al.1991, Biochem. Biophys. Res. Comm., 174, 1306; Brookes et al. 1995,Archs. Oral Biol., 40, 1). Examples of amino acid sequences of TRAP,LRAP and a recombinant murine amelogenin rM179 are shown in Table 1 (Tanet al. 1998, J. Dent. Res., 77, 1388). The full-length rM179 isanalogous to the secreted full-length mouse amelogenin M180 lacking onlythe amino-terminal Met1 and phosphorylation of Ser16. Recombinant rM166was engineered to create the amelogenin lacking the hydrophilicC-terminal 12 amino acids (Simmer et al. 1994, Calcif. Tissue Int., 54,312). TABLE 1 Amino acid sequences of amelogenins.(M) PLPPHPGSPGYINLSYEVLTPLKWYQSMI ³⁰ RQPYPSYGYEPMGGW↓LHHQIIPVLSQQHPP⁶⁰             TRAP SHTLQPHHHLPVVPAQQPVAPQQPMMPVPG⁹⁰HHSMTPTQHHQPNIPPSAQQPFQQPFQPQA¹²⁰ IPPQSHQPMQPQSPLHPMQPLAPQPPLPPL¹⁵⁰FSMQPLSPILPELPLEA↓WPATDKTKREEVD ¹⁸⁰                rM166        rM179The amino acid sequence of LRAP is underlined. The arrow above“TRAP” indicates the location of the C′-terminal residue of TRAP; thearrow above “rM166” indicates the C′terminal residue of rM166. rM179consists of the entire sequence shown above, except for the initiatingmethionine (shown in parentheses).

Amelogenin proteins have been found to self-assemble in vitro undersuitable aqueous conditions to form quasi-spherical quaternary aggregatestructures (nanospheres) (Fincham et al. 1995, J. Struct. Biol., 115,50; Moradian-Oldak et al. 2000, J. Struct. Biol., 131, 27). Thesenanosphere structures have been postulated to be of great importance increating an ultrastructural microenvironment for the controlledformation of highly ordered elongated apatite crystals in enamel(Moradian-Oldak 2001, Matrix Biol., 20, 293; Wen et al. 2001, MatrixBiol., 20, 387).

Researchers have attempted to utilize amelogenins to in vitro calcifyingsystems, which allow the crystal growth of apatite or octacalciumphosphate (OCP) grown from supersaturated calcifying solutions (SCSs) atambient temperatures. OCP has been proposed to be a potent precursor fornatural enamel crystallites (Brown 1979, J. Dent. Res. Special Issue,58B, 857). The previously observed effects of amelogenins on thekinetics of apatite crystal growth and the morphology of OCP aredescribed in the following references: Doi et al. 1984, J. Dent. Res.,63, 98; Aoba et al. 1987, Calcif. Tissue Int., 41, 281; Moradian-Oldaket al. 1998, Biopolymers, 46, 225; Hunter et al. 1999, Calcif. TissueInt., 65, 226; Wen et al. 2000, J. Dent. Res., 79, 1902; Iijima et al.2001, J. Crystal Growth, 222, 615; Iijima et al. 2002, J. Dent. Res.,81, 69.

One frequently employed system was seeded crystal growth of apatite inwhich extracted enamel or synthetic hydroxyapatite (HA) crystals weredispersed into SCSs containing 3-68 μg/ml of amelogenins (Aoba et al.1987, Calcif. Tissue Int., 41, 281; Moradian-Oldak et al. 1998,Biopolymers, 46, 225; Doi et al. (Doi et al. 1984, J. Dent. Res., 63,98) concluded that the seeded enamel crystal growth was inhibited mainlyby the central portion of the amelogenin molecule through their study ofa series of bovine amelogenins of different molecular weights from 5,000to 27,000. Aoba et al. (Aoba et al. 1987, Calcif. Tissue Int., 41, 281)and Moradian-Oldak et al. (Moradian-Oldak et al. 1998, Biopolymers, 46,225) observed that the parent or the full-length amelogenin was found tohave a slight inhibitory effect on apatite crystal growth most likelydue to some adsorption affinity of its hydrophilic carboxy-terminalmotif on apatite. The inhibition became insignificant after theproteolytic cleavage of this hydrophilic region. Additionally, rM179appeared to have an adherence effect on growing apatite crystals,presumably through its molecular self-association (Moradian-Oldak et al.1998, Biopolymers, 46, 225).

The coating of the present invention is typically applied in thin layersuniformly across the surface of the substrate. In certain embodiments,e.g., where the substrate to be coated is immersed in theamylogenin-containing calcifying solutions, the method allows for theuniform coating of complex surfaces including porous surfaces andrecessed surfaces. The geometry of the substrate surfaces, which may becrucial to the effectiveness of the substrate upon implantation, shouldnot be affected by immersion coating.

According to one embodiment of the method, the substrate is contactedwith a supersaturated calcifying solution, wherein the solutioncomprises an effective amount of an amelogenin-type protein. An“effective amount” is an amount of amelogenin sufficient tosubstantially modify the growth of hydroxapatite or octacalciumphosphate crystals on the surface of the substrate. In one aspect aconcentration of at least approximately 10 μg/ml amelogenin is used.Alternatively, concentrations greater than 100 μg/ml are used toprofoundly modify the crystals.

The method is applied to a variety of substrates, including silicon,metals, ceramics and polymers. In particular, the method is useful forcoating substrates which are intended for medical implantation, e.g.,bone and dental prostheses. Such substrates are typically fashioned ofstrong biocompatible materials such as titanium metal. The method iscompatible with other metals, including titanium alloy, tantalum,tantalum alloy, stainless steel and cobalt chromium alloy. Otherwell-known biocompatible materials such as ultra high molecular weightpolyethylene, hydroxyapatite, Bioglass and Glass Ceramic A-W, may alsobe used.

In one aspect, the substrate is pre-treated prior to coating. Anytreatment that modifies the surface of the substrate to facilitatecrystal formation is a suitable pre-treatment, e.g., chemically treatingthe metal to form nanopockets. Titanium samples, for example, can becleaned ultrasonically and “etched” prior to immersion in thesupersaturated calcifying solution. Etching roughens the surface oftitanium and facilitates crystal growth. Etching may be accomplished byvarious means including, without limitation, chemical, physical orthermal means. One example of chemical etching involves treatment of ametal, e.g., titanium, with hydrofluoric acid, followed by immersion insodium hydroxide. In some embodiments, the substrate is well rinsedbefore contact with the calcifying solution. This method formsnano-sized pores on the surface of the substrate which facilitate theinduction of calcium phosphate crystal growth. Other substrates, such asBioglass, do not require hydrofluoric acid etching for crystal growth.One skilled in the art will recognize what steps are necessary toprepare the substrate for coating by referring to the literature inwhich the properties of the preferred implantable substrates arewell-known and documented. Such steps are analogous to, and may include,crystal seeding and doping procedures.

The supersaturated calcifying solution of the invention comprises anaqueous solution having a pH in the range of about 5 to about 10, oralternatively a pH in the near-physiological range, e.g., about 6.5 toabout 8. The solution contains at least calcium ions and phosphate ions,but may contain other ions, particularly physiologically relevant ionsincluding, but not limited to, magnesium, sodium, chlorine, sulfate,potassium and carbonate. The pH of the solution may be maintained by abuffer such as Tris or any other chemical which provides effectivebuffering capability in the near-physiological pH range. The coatingprocess is carried out typically at temperatures in the physiologicalrange, e.g., approximately 35 to approximately 40 degrees Celsius, butmay be carried out ambient temperatures from approximately 10 toapproximately 35 degrees. In certain embodiments of the coating method,coating takes place at ambient atmospheric pressure.

Concentrations of ions in the supersaturated calcifying solution are inthe range of, e.g., 100 to 200 mM Na⁺, 3-5 mM K⁺, 1-3 mM Ca²⁺, 100 to250 mM Cl⁻, 1 to 2.5 mM HPO₄ ²⁻, 1-2 mM SO₄ ²⁻, and 1 to 100 mM Tris.The ionic strength of the solution is typically in the range of 100 to200 mM, but useful coating may be achieved concentrations outside thatrange. The solution may be prepared by dissolving analytical gradechemicals, e.g., CaCl₂.2H₂O, Ca(NO₃)₂.4H₂O, Na₂HPO₄.7H₂O, NaCl, KNO₃,KH₂PO₄, in deionized water. One skilled in the art will recognize thatother suitable compounds may be dissolved to achieve the same desiredconcentrations of ions. Other biologically relevant ions, such asmagnesium or silicate, may be added or substituted for the ions listedabove. Growth of the enamel-like biomaterial is primarily dependent onthe presence of calcium and phosphate ions. Purified recombinantamelogenin or native amelogenin are first dissolved at higherconcentrations in a buffered solution, then added to the calcifyingsolution to reach the desired final concentration. In another embodimentof the method, therapeutic agents including antibiotics, growth factors,or anti-inflammatory agents, are added to the supersaturated calcifyingsolution so that the agents are incorporated into the enamel-likecoating.

The substrate, or a desired portion of the substrate, is immersed in thesupersaturated calcifying solution for the length of time needed toproduce the desired amount of enamel-like coating on the surface of thesubstrate. Typically, the substrate will be in contact with the solutionfor at least one hour, and more typically for one to seven days. Thelength of time will vary depending on such recognizable factors as theamount of coating desired, the particular concentrations of ions presentin the calcifying solution, the particular amount of amelogenin present,pecularities of the substrate surface, the solution pH and the ambienttemperature and pressure.

In one embodiment of the coating method, the substrate to be coated isfirst contacted with a supersaturated calcifying solution which issubstantially free of any amelogenin-type protein. As used herein, theterm “substantially free” comprises any concentration that will notaffect crystal formation. In one aspect, amelogenin-like proteins arenot present in the solution, or are present only in insignificantconcentrations (e.g., 1 μg/ml or less). As is apparent to those skilledin the art—the specific amount of protein that can be present withoutaffecting crystal formation varies with the substrate, the solution, thepH, ionic strength and the concentration of protein contained within thesecond contacting solution. After a period of incubation in contact withthis first solution, the substrate is contacted with theamelogenin-containing solution discussed above. The firstamelogenin-free solution may comprise ions and ion concentrationssimilar to the solution containing amelogenin. Typically, the firstamelogenin-free solution will comprise higher concentrations of K⁺(e.g.,between 75 and 200 mM) and NO3- (e.g., between 75 and 200 mM) andapproximately half the concentration of Ca²⁺ and HPO₄ ²⁻. According tothis embodiment, the first incubation continues at 37 degrees Celsiusfor 1 to 24 hours, but typically between 2 and 10 hours or between 3 and5 hours.

The methods described above yield a novel enamel-like biomaterial, theproperties of which are influenced by the presence of theamelogenin-type protein in the calcifying solution. As natural enamel inthe body is characterized by the presence of elongated crystals,practitioners of the method will frequently desire that the syntheticenamel-like coating also comprises elongated crystals. The methodsdisclosed herein are capable of creating enamel-like coatings whichconsist of submicron bundles of elongated apatite crystals with anaverage aspect ratio (length/width) of at least two. In addition,analysis of the reactions shows that the resultant coating comprisesapproximately 1×10⁻⁴% to 10% w/w amelogenin-type protein.

Without further elaboration, it is believed that one skilled in the artcan follow the preceding description and utilize the present inventionto its fullest extent. The following examples of specific embodimentsare, therefore, to be construed as merely illustrative and are notintended to limit the disclosure in any way.

EXAMPLES Example 1 Coating of Titanium with Enamel-Like BiomaterialComprising Amelogenin

Materials and Methods.

Recombinant murine amelogenin rM179 was expressed, purified andcharacterized as previously described (Simmer et al. 1994, Calcif TissueInt, 54, 312-319). The rM 179 protein is analogous to the secretedfull-length mouse amelogenin M180 lacking only the amino-terminal Met1and phosphorylation of Ser16 (Fincham et al. 1993, Biochem Biophys ResComm, 197, 248-255). Bovine serum albumin (BSA) purchased from SigmaChemicals (A-4503, Lot77H0504) was used as a control protein forinhibitory activity, which has been well documented previously (Robinsonet al. 1992, J Dent Res, 71, 1270-1274; Radin et al. 1996, J BiomedMater Res, 30, 273-279). Titanium samples of 10×10×1.3 mm were cut fromthe commercially pure titanium sheet (Titanium Industries, Grade 2, ASTMB265). Two types of SCSs were prepared from analytical-grade CaCl₂.2H₂O,Ca(NO₃)₂.4H₂O, Na₂HPO₄.7H₂O, NaCl, KNO₃, KH₂PO₄ to achieve the ionconcentrations listed in Table 2.

SCSs containing rM179 or BSA were made by dissolving the protein intothe blank SCSs at concentrations ranging from 12.5 to 100 μg/ml.Titanium samples were ultrasonically cleaned in distilled-deionizedwater (DDW), acetone, 70% ethanol solution for 20 min each, and in DDWagain for 10 min. The cleaned samples were etched with 10 ml 10% HFsolution for 30 min, immersed in 40 ml 2 N NaOH solution at 85° C. for 5h and completely rinsed with DDW. Two SCSs (SCS1 and SCS2) were employedfor growing apatite crystals on the chemically modified titanium. The pHof SCS1 was 7.4 at room temperature and that of SCS2 was maintained tobe 7.4 at 37° C. by using a Metrohm 718 pH-STAT during the crystalgrowth experiments. The OCP and apatite crystal growth on titanium wasrespectively achieved through the following two processes. Process I:Immersion of the chemically treated titanium samples in blank, rM179- orBSA-containing SCS1—(20 ml per sample at 37° C. for 1 d). Process II:Pre-incubation of the samples in blank SCS1—(20 ml per sample at 37° C.for 4 h) followed by an immersion in blank, rM179- or BSA-containingSCS2—(40 ml per sample at 37° C. for 3 d).

The protein concentrations of those SCSs containing rM179 and BSA beforeand after the crystal growth experiments were measured using reversephase high performance liquid chromatography (HPLC, Vydac, C4-214TP54column, Separations Group, Hesperia, Calif., USA). All the samples wererinsed with DDW, air-dried, and characterized by means of X-raydiffraction (XRD, Rigaku, Cu Kα radiation at 50 kV/70 mA), scanningelectron microscopy (SEM, Cambridge 360, at 15 kV), and transmissionelectron microscopy (TEM, JOEL, JEM-1200EM) coupled with selected areaelectron diffraction (SAED), as previously described (Wen et al. 2000, JBiomed Mater Res, 52, 762-773).

Results.

SEM micrographs of an untreated (as received) titanium surface, andthose after HF and NaOH treatment are shown in FIG. 1. It was noted thatthe HF etching increased the roughness of titanium surface for theemerging of nanosized structures (FIG. 1 b). Nano-sized pores wereformed at the sample surface by the subsequent NaOH immersion (FIG. 1c). The two-step chemical treatment of titanium samples was performed tocreate a bioactive-—Ca—P inductive—surface. All the samples werecompletely coated by a mineral layer after different immersionprocedures as indicated by their representative XRD patterns in FIG. 2.The formation of a nanometer scaled porous oxide layer formed attitanium surface after the chemical treatment was the key to the mineralinitiation from the SCS1 (Wen et al. 1998, J Mater Sci Mater Med, 9,121-128; Wen et al. 1998, J Crystal Growth, 186, 616-623).

As determined by the X-ray diffraction patterns in FIG. 2 a very thinlayer of apatite was nucleated on the sample surface followed by thegrowth of OCP crystals in Process I (FIG. 2 a-c) while apatite was theonly mineral phase developed in Process II (FIG. 2 d-f). Only titaniumpeaks were detected in the pattern of chemically modified titanium (FIG.2 h) but apatite crystals were formed after 4 h immersion in blank SCS1(FIG. 2 g). The XRD patterns of OCP/apatite formed from Process I (FIG.2 a-c) were similar to one another regardless of the presence ofdifferent concentrations of different proteins, so were the XRD patternsof apatite formed from Process II (FIG. 2 d-f).

FIG. 3 represents the SEM micrographs of the cross sections of the Ca—Pcoatings (FIG. 3 a) and the morphology of OCP crystals formed on thechemically modified titanium by process I, in the absence (FIG. 3 b),and presence of amelogenin (FIG. 3 c-d) and albumin (FIG. 3 e-f). After1 day of immersion in blank SCS1 about 15-μm thick OCP crystal layer wasprecipitated on titanium. The crystals were seen in the characteristicplated-like shape of OCP and measured ˜200 nm thick and several micronsacross (FIG. 3 b). The application of rM179 showed no significantinhibitory effect on either the morphology or sizes of OCP crystals overthe concentration range of 12.5-100 μg/ml (FIG. 3 c-d), whereas thepresence of BSA significantly altered the plated-like OCP crystals intoa round edged, curved shape indicating a general inhibitory effect (FIG.3 e-f).

FIG. 4 is the SEM micrographs of apatite crystals grown by process II inthe absence (FIG. 4 a-d) and the presence of amelogenin (FIG. 4 e-f) andalbumin (FIG. 4 g-h). The apatite crystals developed from Process IIwithout applying any proteins were observed under SEM to be in aslightly curved platy shape, mostly less than 2 μm across (FIG. 4 a-d).Regardless of the concentration applied, BSA showed dramatic inhibitionas indicated by the significantly reduced crystal sizes (FIG. 4 g-h).Interestingly, the effects of rM179 appeared to be dose dependent. Nosignificant effect was observed at a concentration of 50 μg/ml (FIG. 4e). However, at higher concentration, e.g., 100 μg/ml, amelogeninremarkably modulated the plated-like crystals into submicron-sizedstructures (FIG. 4 f). These were characterized by TEM in combined withSAED to be bundles of elongated apatite crystals with a preferentialorientation of 002 (c-axis) (FIG. 5).

Table 3 summarizes the consumption rate (%) of recombinant amelogeninrM179 and BSA during OCP and apatite crystal growth on titanium surface.rM179 is consumed during the OCP and apatite crystal growth verydifferently from BSA. There was more BSA absorbed by OCP crystals thanapatite crystals while much more rM179 was incorporated into the apatitethan OCP crystal layers. TABLE 2 Concentrations (mM) of ions present inthe PBS and two SCSs employed for Ca—P crystal growth on biomaterials.Ion SCS1 SCS2 Na⁺ 136.8 — K⁺ 3.71 144.6 Ca²⁺ 3.10 1.5 Cl⁻ 185.5 — HPO₄²⁻ 1.86 0.9 NO₃ ⁻ — 145.8 Tris 50

TABLE 3 Consumptions (%) of rM179 and BSA during OCP and apatite crystalgrowth on titanium analyzed by HPLC. Protein OCP Apatite rM179-50 μg/ml27 97.5 rM179-100 μg/ml 22.5 78 BSA-50 μg/ml 32 7 BSA-100 μg/ml 25 9

Example 2 Coating of Bioglass® with Enamel-Like Material ComprisingAmelogenin

Materials and Methods.

Bioactive (Ca—P inducible) materials may serve as the substrates forapatite crystal growth from SCSs. One of these advanced biomaterials isbioactive glass, which induces apatite formation by immersion in abuffer solution of Tris-HCl or phosphate buffered saline (PBS) (Zhong etal. 1997, Transactions of the 23rd Annual Meeting of the Society forBiomaterials, 125). 45S5 type Bioglass® discs were provided byUSBiomaterials Corporation. This material has been clinically appliedfor over 7 years as bone graft materials, especially in periodontaldefect repair (Hench 1994, Bioceramics 7, Ö. H. Andersson, and A.Yli-Urpo (Eds.), Butterworth-Heinemann Ltd., Oxford, 3; Hench et al.1996, Life Chem. Rep., 13, 187).

A PBS and two SCSs (SCS1 and SCS2) were employed for growing apatitecrystals on the Bioglass discs. The detailed compositions of PBS, SCS1and SCS2 are listed in Table 4. The pH of both PBS and SCS1 were 7.4 atroom temperature and that of SCS2 were always maintained to be 7.4 at37° C. The rM179 and rP172 proteins were prepared as previouslydescribed by Simmer et al. (Simmer et al. 1994, Calcif. Tissue Int., 54,312) and Ryu et al. (Ryu et al. 1999, J. Dent. Res., 78, 743). Bovineserum albumin (BSA) was used as a control protein for inhibitoryactivity, which has been well documented previously (Radin et al. 1996,J. Biomed. Mater. Res., 30, 273; Gilman et al. 1994, J. Inorganic.Biochem., 55, 3142-44). The concentration applied in the SCSs for allthe proteins was 50 μg/ml. The apatite crystal growth on Bioglass wasachieved in the following two ways: (1) Direct Immersion: Immersing thesamples in blank and rP172-containing SCS1 (SCS1_(b) and SCS1_(rM172))at 37° C. for 0.5, 1, 2 or 4 h; (2) PBS Pre-incubation: Incubating thesamples in PBS at 37° C. for 1 week followed by immersions in blank,BSA- or rM179-containing SCS2 (SCS2_(b), SCS2_(BSA) and SCS2_(rM179)) at37° C. for 3 d. TABLE 4 Concentrations (mM) of ions present in the PBSand two SCSs employed for Ca—P crystal growth on biomaterials Ion PBSSCS1 SCS2 Na⁺ 157.2 136.8 — K⁺ 4.44 3.71 144.6 Ca²⁺ — 3.10 1.5 Cl⁻ 139.6185.5 — HPO₄ ²⁻ 11.9 1.86 0.9 NO₃ ⁻ — — 145.8 Tris — 50 —

Direct Immersion.

The surface transformation of Bioglass observed in SCS1_(b) was in agood consistence with the general reaction sequence occurred atbioactive glass surfaces during implantation or in vitro immersion(Hench 1998, J. Am. Ceram. Soc., 81, 1705). The smooth sample surface asimaged by atomic force microcopy (AFM) (FIG. 6A) was changed to be veryrough after 0.5 h of immersion because of the glass network dissolution.Spherical silica-gel particles with diameters of 150-300 nm consistingof substructures of 20-60 nm across were formed after 1 h of immersion.The chemisorption of amorphous Ca—P and crystallization of nanophaseapatite occurred epitaxially on the silica-gel structures during 1-4 hof immersion. The presence of rP172 dramatically modulated the Bioglasssurface reaction during SCS1_(rP172) immersion. In the first 0.5 h ofimmersion, more than 95% of rP172 protein in solution was adsorbed ontothe sample surfaces as determined by analytical reverse phase highperformance liquid chromatography (HPLC). It was indicated in FIG. 6Bthat the protein self-assembled into spherical assemblies of 10-60 nm indiameters. During 0.5-4 h of SCS1_(rp172) immersion, the proteinassemblies of rP172 remarkably induced the formation of orientatedsilica-gel plates (about 100 nm wide and 50 nm thick) and subsequentlyof platy apatite minerals (FIG. 6D), which were obviously different fromthose formed in SCS1_(b) (FIG. 6C). Under TEM, the apatites grown after2-4 h of SCS1_(b) immersion were revealed to be rod crystals thatmeasured about 100 nm thick and 500 nm long. However, it appeared thatthe crystals formed after 2-4 h of SCS1_(rP172) immersion all adopted anelongated shape. They were in a length comparable to the crystals formedin SCS1_(b) but significantly reduced thickness only about 5-7 nm. Thehighly organized long and thin crystals observed after 4 h ofSCS1_(rP172) immersion strikingly resembled the apatite crystalsobserved in the early stage of enamel biomineralization (Fincham et al.1995, J. Struct. Biol., 115, 50; Diekwisch et al. 1995, Cell TissueRes., 279, 149).

PBS Pre-Incubation.

Mineral layers precipitated on all the treated samples werecharacterized to be apatite by X-ray diffraction (XRD) and Fouriertransmission infrared spectroscopy (FTIR). FIG. 6 presents scanningelectron microscopy (SEM) images of the apatites formed after differentimmersions. Plate-shaped crystals (˜50 nm thick and 300-600 nm across)were observed on the samples after PBS incubation. The crystals grownfrom SCS2_(b) were of the typical plate shape except for a slightincreased thickness, while needle-shaped crystals (200-300 nm long and50-70 nm thick) were precipitated on the SCS2_(BSA)-immersed samples. Itwas surprising to observe that the apatites deposited on theSCS2_(rM)179-immersed samples adopted an elongated, curved shape (˜500nm long and ˜120 nm thick). They were revealed by transmission electronmicroscopy (TEM) to be bundles of lengthwise crystals (15-20 nm thick)orientated parallel to one another, much alike the long and thincrystals observed in the very early stage of tooth enamel formation(Fincham et al. 1995, J. Struct. Biol., 115, 50; Diekwisch et al. 1995,Cell Tissue Res., 279, 149). The modulating effects of rM179 on apatitecrystals are distinctly different from the overall inhibition of BSA.

Atomic force microscopic study has revealed a progressive accretion ofrM179 molecules during nanospheres assembly in a Tris-HCl buffer atconcentrations from 12.5 to 300 μg/ml (Wen et al. 2001, Matrix Biol, 20,387-35). At low concentrations (12.5-50 μg/ml), nanospheres withdiameters varying from 7 to 53 nm were identified while atconcentrations between 100-300 μg/ml the size distribution wassignificantly narrowed so that nanosphere diameters were consistentlybetween 10 and 25 nm. These nanospheres were observed to be the basicbuilding blocks of both engineered rM179 gels and the developing enamelextracellular matrix. We infer that the stable 15-20 nm nanospherestructures generated in the presence of high concentrations ofamelogenins may be of great importance in creating a highly organizedultrastructural microenvironment required for the formation of initialenamel apatite crystallites or synthesizing materials having enamel-likestructures.

1-26. (canceled)
 27. An implantable article, comprising: a biocompatiblesubstrate; and an enamel-like surface coating chemically bonded to atleast a portion of the substrate, the coating comprising apatitecrystals and an amelogenin-type protein, wherein said crystals are lessthan a 1 μm in length and have on average an aspect ratio ofapproximately two or greater.
 28. The article of claim 27, wherein thecoating comprises ions selected from the group consisting of calcium,phosphate, sodium, sulfate, magnesium, chlorine, silicate, and mixturesthereof.
 29. The article of claim 27, further comprising an effectiveamount of a therapeutic agent.
 30. A method for enhancing bone ingrowthor soft tissue attachment comprising implanting the article of claim 27onto a bone surface or soft tissue.
 31. (canceled)